Immunoassay method for lysed whole blood

ABSTRACT

An immunoassay method in which blood can be measured even without pretreatment by a centrifuge etc. In the present invention, antibodies or antigens in a sample are subjected to agglutination reaction with insoluble carriers onto which antigens or antibodies specifically reacting with the antibodies or antigens in the sample have been imrobilized and the resulting agglutination mixture is determined for the change in its absorbance or in its scattered light by irradiation with light, wherein said sample is whole blood and the whole blood is forcibly lysed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an immunoassay method and in particular to an immunoassay method for use with lysed whole blood in which antibodies or antigens in a sample are subjected to agglutination reaction with insoluble carriers onto which antigens or antibodies specifically reacting with the antibodies or antigens in the sample have been immobilized. The resulting agglutination mixture is irradiated with near infrared or infrared rays to determine its change in absorbance or its change in scattered light.

2. Description of Related Art

Japanese Patent Publication No. 11575/1983 discloses a prior art method which comprises antigen-antibody reaction between antigen- or antibody-immobilized insoluble carriers and antibodies or antigens in a humor sample, then irradiating the reaction mixture with light with a wavelength of 600 to 2400 nm and measuring the increase in its absorbance. By virtue of its usefuiness, this method has become the mainstream of immunoassay method at present as a so-called latex immunoturbidimetry.

However, the measurement sample used in said measurement method is water, serum, urine, saline etc. In addition, matters that require attention in general blood taking for clinical examination are that hemolysis should be avoided to a maximum degree and blood should be separated into serum and plasma as rapidly as possible. The reasons for this include the effect of hemolysis on optical measurement, the incoming and outgoing of substances such as Na, K, Cl through blood membrane, the effect of movement by blood corpuscles metabolism (i.e., transfer of lactic acid and pyruvic acid to serum by glycolysis) and the effect of the difference in concentration of the object component in blood corpuscles and in serum.

For the above reasons, blood obtained from a subject should be a sample separated into serum or plasma by centrifugation. Therefore, such pretreatment by centrifugation may not be carried out in small or private laboratories or urgent laboratories other than central laboratories in large or middle hospitals where a large amount of blood can be dealt with, and therefore the above method is not necessarily universal.

Under the circumstances of such general whole blood handling, in the field of clinical examination there is no accurate and quantitative measurement method in which whole blood can be directly used as a measurement sample without separating it into serum and plasma. Further, the measurement of blood using optical means without hemolysis is inappropriate because of high turbidity caused by erytlirocyte.

OBJECTS AND SUMMARY OF THE INVENTION

In view of the foregoing, the present invention was made, and a first object is to provide an immunoassay method which can be effected easily in a short time even without pretreating blood by, e.g., a centrifuge, and a second object is to provide an immunoassay method using whole blood directly as a sample in which blood corpuscles are subjected intentionally to forcible hemolysis in a manner not to affect immunoreaction so that accurate data can be obtained in combination with various quantitative measurement reagents.

As a result of their research, the present inventors unexpectedly found that antigens or antibodies in whole blood can be determined by subjecting whole blood intentionally to forcible hemolysis in a manner not to affect agglutination reaction, as opposed to the fixed concept of general blood taldng for clinical examination, that is, hemolysis should be avoided to a maximum degree and blood should be separated into serum and plasma as rapidly as possible.

To achieve the first object, the present invention comprises an immunoassay method in which antibodies or antigens in a sample are subjected to agglutination reaction with insoluble carriers onto which antigens or antibodies specifically reacting with the antibodies or antigens in the sample have been immobilized and the resulting agglutination mixture is determined for the change in its absorbance or in its scattered light by irradiation with light, wherein said sample is whole blood and the whole blood is forcibly lysed.

In this case, the means of forcible hemolysis can include:

(1) mixing whole blood with a low osmotic solution;

(2) mixing blood with a solution of saponins for hemolysis;

(3) freezing and thawing whole blood; and

(4) ultrasonicating whole blood.

Alternatively, saponins for hemolysis may be incorporated into an insoluble particle suspension reagent onto which antibody or antigen specifically reacting with antigen or antibody has been immobilized.

To achieve the second object, the immunoassay method according comprises the step of subjecting antibodies or antigens in whole blood as a sample to agglutination reaction with an insoluble particle suspension reagent onto which antigens or antibodies specifically reacting with the antibodies or antigens in the whole blood have been immobilized, the step of determining the resulting agglutination mixture for the change in its absorbance or in its scattered light by irradiation with light, and the step of calculating the hematocrit % of as follows by the sample:

    A'=A×100/(100-hematocrit %)

where A is the absorbance or its change or the strength of light scattering or its change actually determined, and A' is the corrected absorbance or its change or the strength of light scattering or its change assuming that the plasma component in the sample is 100%.

According to the first object, the following effects are achieved:

(1) By using a whole blood sample directly in the measurement procedure without subjecting it to pretreatment such as centrifugation etc., measurement time can be shortened, measurement costs can be reduced, and a measurement operation can be simplified. Because centrifugation is not required, the costs for a centrifuge or centrifuge tube, the operation for transferring a sample to a centrifuge tube, the time for centrifugation can be eliminated, and the opportunity for an operator to come into contact with blood can be decreased whereby the danger of infection can be significantly reduced.

(2) By subjecting blood corpuscles in whole blood to forcible hemolysis in a manner not to cause any effect on antigen-antibody reaction, the method can be combined with a measurement kit using general latex immunoturbidimetry whereby accurate measurement data can be obtain while a wide variety of applications is made feasible.

(3) By incorporating a hemolysis reagent into a latex reagent, the constitution of the measurement apparatus can be simplified and the measurement time can be reduced.

According to the present invention, accurate data can be obtained by hematocrit correction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing an example of the cell used in the method of the present invention.

FIG. 2 is a schematic drawing showing the constitution of the spectrophotometer used in the method of the present invention.

FIG. 3 is a drawing showing an example of the freezing cell holder used in the method of the present invention.

FIG. 4 is a drawing showing an example of the ultrasonication nozzle used in the method of the present invention.

FIG. 5 is an absorption spectrum at 300 to 1000 nm of whole blood having been subjected to hemolysis with aqueous solutions of various hemolysis reagents

FIG. 6 is a drawing showing hemolysism reaction time course at 800 nm of whole blood having been subjected to hemolysis with aqueous solutions of various hemolysis reagents.

FIG. 7 is a calibration curve of change in absorbance per minute as a function of CRP concentration obtained when CRP measurement is carried out.

FIG. 8 is a graph showing a correlation between a whole blood sample and a plasma sample when no hematocrit correction is carried out.

FIG. 9 is a graph showing a correlation between a whole blood sample and a plasma sample when hematocrit correction is carried out.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention is described in detail by reference to Examples.

The reagents examined in the Example are shown in Table 1 below. The symbols "a" to "g" in Table, 1 are identical with those in FIGS. 5 to 7.

                                      TABLE 1                                      __________________________________________________________________________                        Content                                                                             Absorbance                                                                           ΔAbsorbance/                                      Hemolysis Method                                                                           (W/V %)*                                                                            (800 nm)                                                                             min                                              __________________________________________________________________________     Hemo-                                                                               a Pure Water       0.204 0.003                                            lysis  (Deionized Water)                                                       Reagent                                                                             b Saponin Aqueous Solution                                                                   0.5  0.147 0.000                                            Aqueous                                                                             c Triton X-100                                                                               0.5  0.146 0.000                                            Solu-  (Non-Ionic Surface Active                                               tion   Agent)                                                                       d Tween-20    0.5  0.298 0.055                                                   (Non-Ionic Surface Active                                                      Agent)                                                                       e Brij-35     0.5  2.312                                                         (Non-Ionic Surface Active                                                      Agent)                                                                       f Sodium Lauryl Sulfate                                                                      0.5  0.176 0.000                                                   (Anionic Surface Active                                                        Agent)                                                                       g Benzalkonium Chloride                                                                      0.5  0.139 0.000                                                   (Cationic Surface Active                                                       Agent)                                                                       h Freezing Hemolysis                                                                              0.163 0.000                                                 i Ultrasonication Nozzle                                                                          0.196 0.001                                                   Hemolysis                                                                    j Physiological Saline                                                                            3.000                                                  __________________________________________________________________________      *W/V % = weight/volume %                                                 

EXAMPLE 1

Hemolysis by Hemolysis Reagents

0.04 ml of human whole blood collected in a usual manner using an EDTA-2K anticoagulant was placed in quartz cell 5 with a cell length of 5 mm as shown in FIG. 1. 2.0 ml of each of Hemolysis regent aqueous solutions "a" to "g" shown in Table 1 was added to it and monitored for its absorption spectrum at 300 to 1000 nm (see FIG. 5) in spectrophotometer 1 (e.g., U-3410, produced by Hitachi, Ltd., Japan), for its Hemolysis reaction time course at 800 nm (see FIG. 6), for its absorbance 5 minutes after the reaction was initiated and for the change in its absorbance at 800 nm for 1 minute between 4 and 5 minutes after the reaction was initiated (see Table 1) to examine the ability of each hemolysis reagent to lyse blood.

In FIG. 2, 2 is a halogen lamp as a light source emitting irradiation light L, e.g., near infrared or infrared rays; 3 is a condensing lens; 4 is a diffraction grating; 6 is an amplifier; and 7 is an arithmetic and recording device such as computer 8 is a display. "S" is whole blood having been subjected to hemolysis treatment as a sample accommodated in cell 5.

As shown in FIG. 5, non-lysed blood treated with the reagent "j" (physiological saline) showed an absorbance of 2.5 or more due to its turbidity at the shown wavelengths, resulting in affecting the optical detection of latex agglutination reaction. On the other hand, it was found that as shown in FIG. 5, such turbidity as described above disappeared by use of the reagent "a" (pure water) and the reagent "b" (saponin aqueous solution) so that the degree of agglutination of latex can be determined. As can be seen from Table 1 and FIG. 6, the reagent "a" (pure water), the reagent "b" (saponin), the reagent "o" (Triton X-100), the reagent "f" (sodium lauryl sulfate), and reagent "g" (benzalkonium chloride) have the ability to lyse whole blood in a short time.

EXAMPLE 2

Hemolysis by Freezing

FIG. 3 shows one embodiment of freezing cell holder 9 for use in lyzing whole blood, which can receive and maintain cell 5 in it and includes a Peltier element 12 (a product of, e.g., Merukoa Co., Ltd.) attached to cell block 11 made of aluminum provided with photometric window 10. 13 is a power source for applying a direct current as necessary to Peltier element 12, and L is a near infrared or infrared ray from the power source 2.

0.04 ml of human whole blood collected in a usual manner using an EDTA-2K anticoagulant was accommodated in cell 5 placed in freezing cell holder 9 as shown in FIG. 3, and the human whole blood was completely frozen by applying an electric current to the Peltier element 12 for 10 minutes in a predetermined direction. Thereafter, the frozen human whole blood was thawed by applying an electric current in the reverse direction to the Peltier element 12, then diluted with 2.0 ml physiological saline, and examined for its absorbance 5 minutes after the reaction was initiated and for the change in its absorbance at 800 nm for 1 minute between 4 and 5 minutes after the reaction was initiated (see Table 1) to determine the ability of the hemolysis reagent to lyse blood. As can be seen in "h" in Table 1, human whole blood can be lysed by freezing and thawing.

EXAMPLE 3

Hemolysis by Ultrasonication

FIG. 4 shows one embodiment of ultrasonic nozzle 14 for use in lysing whole blood, which includes ultrasonic radiator 16 attached to stainless steel nozzle 15, and 17 is a radiator circuit, and 18 is a suction syringe. 0.04 ml of human whole blood obtained in a usual manner using an EDTA-2K anticoagulant was suctioned into nozzle 15, and the ultrasonic radiator 16 was run for 5 minutes to lyze human whole blood B in nozzle 15. Thereafter, the lysed human whole blood B was accommodated in nozzle 15, then diluted with 2.0 ml physiological saline, and examined in spectrophotometer 1 for its absorption at 800 nm 5 minutes after the reaction was initiated and for the change in its absorbance for 1 minute between 4 to 5 minutes after the reaction was initiated (see Table 1) to determine the ability of the hemolysis reagent to lyse blood. As can be seen from symbol "i" in Table 1, human whole blood can be lysed by ultrasonicating it.

EXAMPLE 4

CRP Measurement Method 1

1) Preparation of Anti-CRP Antibody Sensitized Latex Solution

An about 10 mg/ml anti-human CRP (C-reactive protein rabbit antibody solution (pH 7.5, 100 mmol/1 Tris-HCl buffer, 0.1% sodium amide) was added to 10 ml polystyrene latex with an average particle diameter of 0.2 μm (produced by, e.g., Japan Synthetic Rubber Co., Ltd., Japan; 10% solid content), and the mixture was allowed to stand at 30° C. one whole day and night and then centrifuged at 3600 rpm to give precipitates. 0.2 W/V % bovine serum albumin, pH 8.5, 100 mmol/l Tris-HCl buffer was added to the precipitates to prepare an anti-CRP antibody sensitized latex solution.

2) CRP Measurement Method

0.04 ml of human whole blood collected in a usual manner using an EDTA-2K anticoagulant was placed in cell 1, and 0.5 ml of each of hemolysis regent aqueous solutions "a" to "g" shown in Table 1 was added to it, and the mixture was incubated at 37° C. for 3 minutes, and 1.5 ml of the anti-human CRP antibody sensitized latex suspension prepared in item 1) above was added to it, and the change in its absorbance at 800 nm for 1 minute between 4 and 5 minutes after the reaction was initiated was determined.

Separately, a calibration curve of the above sample was prepared using a commercial latex inmmunoturbidimetry CRP measurement kit intended for use in examining plasma as its sample. FIG. 3 shows a calibration curve prepared using the results obtained in the above CRP measurement, and a calibration curve excellent in sensitivity as shown in symbols "a" and "b" in the figure was obtained using whole blood lysed forcibly with pure water "an, saponin aqueous solution "b" etc. However, the results indicated that surface active agents "c" to "g" inhibit agglutination reaction and are thus not suitable for immunoreaction, as shown in the symbols "c" to "g" in the figure.

EXAMPLE 5

CRP Measurement Method Using Blood Sample Lysed by Freezing or Ultrasonication

The operation of dilution with physiological saline after hemolysis in Example 2 or 3 was replaced by addition of 2.0 ml of the anti-human CRP antibody sensitized latex suspension prepared in EXAMPLE 4, and a change in absorbance at 800 nm for 1 minute between 4 to 5 minutes after the reaction was initiated was determined in spectrophotometer 1.

Separately, a calibration curve of the above sample was prepared using a commercial latex immunoturbidimetry CRP measurement kit intended for use in examining serum or plasma as its sample. As shown in symbols "h" and "i" in FIG. 7, a calibration curve excellent in sensitivity was obtained.

EXAMPLE 6

CRP Measurement Method 2.

The same measurement method as in Example 4 was used except that a commercial latex immunoturbidimetiy CRP measurement kit was used in place of the anti-human CRP antibody sensitized latex suspension used in Example 4 and 0.5 w/v S saponin aqueous solution was used as a hemolysis reagent in order to determine a change in absorbance at 800 nm for 1 minute between 4 to 5 minutes after the reaction was initiated. A comparison between a calibration curve (n=40) using measurement values of whole blood as a sample determined according to the present invention and a calibration curve using measurement values of serum as a sample determined according to a general method indicated good correlation as shown in FIG. 8.

EXAMPLE 7

Hematocrit Correction

To correct the measurement values obtained according to the present invention in Example 6, the whole blood was simultaneously determined for its hematocrit value by means of an erythrocyte counter (e.g., LC-240A manufactured by Horiba Seisakusho K.K., Japan) using the following formula (1):

    A'=A×100/(100-hematocrit %)                          (1)

where A is the actually determined change in absorbance, and A' is its corrected change assuming that the plasma component in the sample is 100%. A comparison (n=40) between the measurement values thus corrected and the measurement values determined in a general method using serum as a sample indicated further improved correlation as compared with that of Example 6, as shown in FIG. 9.

In the Examples above, the change in absorbance of an agglutination mixture by light irradiation was determined; alternatively, the change in scattered light may be determined.

The present invention is practiced in the embodiments described above and demonstrates the following effects:

According to the present first invention, measurement time can be shortened, measurement costs can be reduced, and measurement operation can be simplified because a whole blood sample can be used directly without subjecting it to pretreatment such as centrifugation, etc. Further, the opportunity for an operator to come into contact with blood can be decreased and the danger of infection can be significantly reduced.

According to the present second invention, accurate data can be obtained by conducting hematocrit correction. 

What is claimed is:
 1. An immunoassay method in which whole blood is lysed and antigens in the sample of whole blood are reacted in an agglutination reaction with insoluble carriers onto which antibodies specifically reacting with the antigens in the sample have been immobilized,comprising the steps of:lysing the whole blood with a hemolysis reagent; irradiating the resulting agglutination mixture with light including a wavelength substantially free from absorption by hemoglobin and the hemolysis reagent; and measuring the resulting agglutinating mixture to determine a change in absorbance by the irradiation of the reacted agglutination mixture with the light of the wavelength substantially free from absorption of the hemoglobin and the hemolysis reagent, where said sample is whole blood and the whole blood is lysed at or before the agglutination reaction.
 2. The immunoassay method according to claim 1, wherein the whole blood is lysed by mixing the whole blood with an pure deionized later.
 3. The immunoassay method according to claim 1, wherein the whole blood is forcibly lysed by mixing the whole blood with a solution of saponins for hemolysis.
 4. The immunoassay method according to claim 1, wherein saponins are added into a reagent formed by suspending insoluble carriers in a buffer solution onto which the antibodies specifically reacting with antigens in the whole blood sample have been immobilized.
 5. An immunoassay method comprising the steps of:lysing a whole blood sample with a hemolysis reagent; reacting antigens in the hemolysed blood sample in an agglutination reaction with antibodies immobilized onto insoluble carriers wherein the antibodies are specific to the antigens in the hemolysed blood sample; measuring the resulting agglutination mixture for a change in absorbance by irradiation with light of a wavelength substantially free from absorption by both hemoglobin and the hemolysis reagent; determining a hematocrit percent of the sample; and calculating a plasma concentration with the hematocrit percent by the following equation:

    A'=A×100/(100-hematocrit %)

where A is the absorbance, and A' is the corrected absorbance assuming that a plasma component in the sample is 100%.
 6. A immunoassay method of quantifying antigens in a sample of whole blood, comprising the steps of:providing the sample of the whole blood; hemolyzing the whole blood sample with a hemolysis reagent to hemolyze the blood corpuscles; reacting the hemolysed whole blood sample in an agglutination reaction to form a reaction mixture wherein a predetermined antigen in the hemolysed whole blood sample specifically reacts with an antibody immobilized onto an insoluble carrier; irradiating the reaction mixture in the sample with irradiation in the wavelength range of approximately 800 nm which is substantially free from absorption by both hemoglobin and the hemolysis reagent; and measuring in the wavelength range of approximately 800 nm an absorbance of the incident radiation through the reaction mixture to determine the quantity of antigens in the sample.
 7. The immunoassay method of claim 6, wherein the step of hemolysis is performed by mixing saponin aqueous solution with the blood corpuscles.
 8. A immunoassay method of quantifying a predetermined antigen in a sample of whole blood, comprising the steps of:providing the sample of the whole blood; adding a hemolysis reagent to provide a hemolysed blood sample; adding a latex reagent containing insoluble carriers with antibodies immobilized thereto; reacting antigens in the hemolysed blood sample in an agglutination reaction to form a reaction mixture with said antibodies immobilized onto insoluble carriers in the latex reagent irradiating the reaction mixture in the sample with irradiation which includes a wavelength range which is substantially free from absorption by both hemoglobin and the hemolysis reagent; and measuring the resulting reaction mixture only in the wavelength range which is substantially free from absorption by both hemoglobin and the hemolysis reagent, an absorbance of the incident radiation through the reaction mixture to determine the quantity of antigens in the sample by determining a hematocrit percentage of the sample; and calculating a plasma concentration with the hematocrit percent by the following equation:

    A'=A×100/(100-hematocrit %)

where A is the absorbance, and A' is the corrected absorbance assuming that a plasma component in the sample is 100%.
 9. The immunoassay method of claim 8, wherein the step of hemolysis is performed with a saponin aqueous solution.
 10. The immunoassay method of claim 9, wherein the measuring and calculating step is performed with the use of an erythrocyte counter. 